Beta coronavirus temperature-sensitive strains and vaccines

Abstract

Provided is a strain that is effective as an active ingredient of a vaccine against a betacoronavirus. This SARS-CoV-2 virus includes a non-structural protein that has the following responsible mutation(s): a mutation in the amino acid residue corresponding to the L of position 445 of SEQ ID NO: 1 in NSP3; a mutation in the amino acid residues corresponding to the G of position 248 and the G of position 416 of SEQ ID NO: 2 in NSP14; and / or a mutation in the amino acid residue corresponding to the V of position 67 of SEQ ID NO: 3 in NSP16.

Description

[Technical Field]

[0001] This invention relates to a temperature-sensitive strain of betacoronavirus and a vaccine using the same. [Background technology]

[0002] The infectious disease (COVID-19) caused by the novel coronavirus (SARS-CoV-2), which originated in Wuhan, China in 2019, has caused a global pandemic and become a major social problem. As a result, vaccine development against SARS-CoV-2 is progressing rapidly worldwide. As of October 2020, the only approved vaccine is Sputnik V, approved in Russia (Non-Patent Literature 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] THE LANCET, VOLUME 396, ISSUE 10255, P887-897, SEPTEMBER 26, 2020 [Overview of the project] [Problems that the invention aims to solve]

[0004] However, Sputnik V is still in clinical trials, and questions have been raised about its safety and effectiveness. Furthermore, even if vaccination begins, there is still a risk of it being ineffective or causing serious side effects, and it remains unclear whether it will be a decisive solution to contain the epidemic. Therefore, more options are needed for a vaccine against the SARS-CoV-2 virus.

[0005] Therefore, the present invention aims to provide a strain that is effective as an active ingredient in a vaccine against the SARS-CoV-2 virus. Furthermore, since there may be other viruses besides the SARS-CoV-2 virus among betacoronaviruses like the SARS-CoV-2 virus, the present invention also aims to provide a strain that is effective as an active ingredient in a vaccine against betacoronaviruses in general. [Means for solving the problem]

[0006] As a result of diligent research, the inventors discovered that a specific mutant strain of the SARS-CoV-2 virus exhibits reduced replication at human body temperature (so-called lower respiratory tract temperature). Furthermore, by conducting a reverse mutation test on this specific mutant strain, they found that a specific causative mutation can cause reduced replication at human body temperature (so-called lower respiratory tract temperature) in betacoronaviruses in general. This invention was completed by further research based on this finding.

[0007] In this invention, "temperature sensitivity" refers to the property of having the ability to grow specifically at low temperatures (i.e., the temperature of the human upper respiratory tract), and is achieved by acquiring the characteristic that the ability to grow at high temperatures (i.e., the temperature of the human lower respiratory tract) is limited. In this specification, the term "low-temperature acclimatization" is used to mean acquiring the property of having the ability to grow specifically at low temperatures (i.e., the temperature of the human upper respiratory tract), and in reality, this is achieved by acquiring the characteristic that the ability to grow at high temperatures (i.e., the temperature of the human lower respiratory tract) is limited, so it is used to mean "temperature-sensitive preparation." Furthermore, a strain that has been "temperature-sensitive" is called a "temperature-sensitive strain." For this reason, in this specification, "low-temperature acclimatized strain" and "temperature-sensitive strain" are synonymous. In addition, in this invention, "responsible mutation" refers to a mutation that can cause a mutant phenotype (a phenotype that expresses the traits of an organism has changed due to a mutation) or has a causal relationship with a mutant phenotype, and "responsible mutation for temperature sensitivity" refers to a mutation that can cause the acquisition of temperature sensitivity or has a causal relationship with the acquisition of temperature sensitivity. The present invention provides inventions in the following embodiments.

[0008] Item 1. Betacoronavirus temperature-sensitive strains containing a non-structural protein having the following mutations: (b), combinations of (e) and (f), and / or (h) as the mutation responsible for temperature sensitivity: (b) A mutation in the amino acid residue corresponding to leucine at position 445 of the amino acid sequence shown in SEQ ID NO: 1 in NSP3, (e) A mutation in the amino acid residue corresponding to glycine at position 248 of the amino acid sequence shown in SEQ ID NO: 2 in NSP14. (f) A mutation in the amino acid residue corresponding to glycine at position 416 of the amino acid sequence shown in SEQ ID NO: 2 in NSP14. (h) A mutation in the amino acid residue corresponding to valine at position 67 of the amino acid sequence shown in SEQ ID NO: 3 in NSP16.

[0009] The following inventions are specific examples of the invention described in item 1 above. Betacoronavirus temperature-sensitive strains comprising a non-structural protein consisting of at least one of the following polypeptides: (I), (II), and (III): (I) At least one polypeptide from (I-1) to (I-3) below: (I-1) A polypeptide (NSP3) consisting of an amino acid sequence in which the leucine mutation at position 445 (b') is the causal mutation, as shown in SEQ ID NO: 1, (I-2) A polypeptide (NSP14) consisting of an amino acid sequence in which the glycine at position 248 (e') and the glycine at position 416 (f') are the responsible mutations, as shown in SEQ ID NO: 2, (I-3) A polypeptide (NSP16) consisting of an amino acid sequence in which the valine mutation (h') at position 67 is the causal mutation, as shown in SEQ ID NO: 3; (II) A polypeptide constituting a betacoronavirus that has acquired temperature sensitivity, wherein one or more amino acid residues other than the amino acid residue related to the responsible mutation are substituted, added, inserted, or deleted in the amino acid sequence of the polypeptide in (I) above; (III) A polypeptide that constitutes a beta coronavirus with acquired temperature sensitivity, wherein the sequence identity of the amino acid sequence excluding the amino acid residue related to the responsible mutation in the amino acid sequence of the polypeptide of (I) is 50% or more.

[0010] Item 2. The virus temperature-sensitive strain according to item 1, wherein the beta coronavirus is a SARS-CoV-2 virus. Item 3. The virus temperature-sensitive strain according to item 1 or 2, wherein the growth ability at the lower respiratory tract temperature of humans is reduced compared to the growth ability of a beta coronavirus containing a non-structural protein without the responsible mutation. Item 4. The virus temperature-sensitive strain according to item 3, wherein the lower respiratory tract temperature of humans is 36 - 38°C. Item 5. The virus temperature-sensitive strain according to any one of items 1 - 4, wherein the mutation of (b) is a substitution to phenylalanine, the mutation of (e) is a substitution to valine, the mutation of (f) is a substitution to serine, and the mutation of (h) is a substitution to isoleucine. Item 6. In the amino acid sequence shown in SEQ ID NO: 1, the NSP3 having the mutation of (b), In the amino acid sequence shown in SEQ ID NO: 2, the NSP14 having the mutation of (e) and the mutation of (f), and / or The virus temperature-sensitive strain according to any one of items 1 - 5, comprising NSP16 having the mutation of (h) in the amino acid sequence shown in SEQ ID NO: 3. Item 7. The virus temperature-sensitive strain according to any one of items 1 - 6, having the mutation of (e) and the mutation of (f). Item 8. The virus temperature-sensitive strain according to any one of items 1 - 6, having the mutation of (h). Item 9. The virus temperature sensitivity according to any one of items 1 - 6, having the mutation of (b). Item 10. A live attenuated vaccine comprising the virus temperature-sensitive strain according to any one of items 1 - 9. Item 11. The live attenuated vaccine according to claim 10, which is administered nasally. Item 12. The live attenuated vaccine according to Item 10, which is administered intramuscularly, subcutaneously or intradermally. Item 13. A beta coronavirus gene vaccine comprising a gene encoding a non-structural protein having, as the responsible mutation for temperature sensitivity, the mutation of (b) below, the combination of the mutations of (e) and (f), and / or the mutation of (h): (b) A mutation of the amino acid residue corresponding to leucine at position 445 of the amino acid sequence shown in SEQ ID NO: 1 in NSP3, (e) A mutation of the amino acid residue corresponding to glycine at position 248 of the amino acid sequence shown in SEQ ID NO: 2 in NSP14, (f) A mutation of the amino acid residue corresponding to glycine at position 416 of the amino acid sequence shown in SEQ ID NO: 2 in NSP14, (h) A mutation of the amino acid residue corresponding to valine at position 67 of the amino acid sequence shown in SEQ ID NO: 3 in NSP16. Item 14. The gene vaccine according to Item 13, which is administered intranasally, intramuscularly, subcutaneously or intradermally.

Advantages of the Invention

[0011] According to the present invention, a strain effective as an active ingredient of a vaccine against beta coronavirus is provided.

Brief Description of the Drawings

[0012] [Figure 1] Shows a method for temperature sensitization (cold adaptation) of SARS-CoV-2. [Figure 2] Shows the confirmation results (CPE images) of the temperature sensitivity (cold adaptation) of SARS-CoV-2. [Figure 3A] Shows the mutation analysis results of each virus strain. [Figure 3B] Shows the CPE images by strains that may have reverted mutations of the temperature-sensitive strain (cold-adapted strain) (A50-18). [Figure 3C] Shows the CPE images by strains that may have reverted mutations of the temperature-sensitive strain (cold-adapted strain) (A50-18). [Figure 3D]This shows the results of confirming the temperature sensitivity of recombinant viruses into which mutations have been introduced in the temperature-sensitive strain (cold-acclimatized strain) (A50-18). [Figure 3E] This shows the results of confirming the temperature sensitivity of recombinant viruses into which mutations have been introduced in the temperature-sensitive strain (cold-acclimatized strain) (A50-18). [Figure 4A] The results of the proliferation analysis of the temperature-sensitive strain (cold-acclimatized strain) (A50-18) are shown. [Figure 4B] The results of the proliferation analysis of the temperature-sensitive strain (cold-acclimatized strain) (A50-18) are shown. [Figure 5] This shows the weight fluctuations of hamsters infected with SARS-CoV-2. [Figure 6] This shows the weight fluctuations of hamsters infected with SARS-CoV-2. [Figure 7] This indicates the amount of virus in the lungs or nasal lavage fluid. [Figure 8] This image shows the lungs of a hamster infected with SARS-CoV-2. [Figure 9] The results of the lung histological analysis of SARS-CoV-2 infected hamsters are shown. [Figure 10] This shows the histological analysis of the lungs of a SARS-CoV-2 infected hamster (HE staining and IHC staining). [Figure 11] This shows the weight fluctuations of hamsters reinfected with SARS-CoV-2. [Figure 12] This shows the weight changes of hamsters after SARS-CoV-2 infection. [Figure 13] This shows the neutralizing antibody titer in the serum of hamsters that recovered from SARS-CoV-2 infection. [Figure 14] This document describes methods for making SARS-CoV-2 more susceptible to cold (low-temperature acclimatization) (G-L50 series). [Figure 15] The results of confirming the temperature sensitivity (cold acclimation) of SARS-CoV-2 (CPE image) are shown. [Figure 16A] The results of mutation analysis of additional isolates (H50-11, L50-33, L50-40) are shown. [Figure 16B]This shows the CPE image of a strain that may have a revert mutation in the temperature-sensitive strain (cold-acclimatized strain) (H50-11). [Figure 16C] This shows CPE images from strains that may have reverse mutations in temperature-sensitive strains (cold-acclimatized strains) (L50-33, L50-40). [Figure 17] This shows the deletions of nucleotide sequences found in temperature-sensitive strains (cold-acclimatized strains) (H50-11, L50-33, L50-40). [Figure 18] Figure 17 shows a schematic diagram of the nucleotide sequence deletion and the corresponding amino acid sequence deletion. [Figure 19] The results of the proliferation analysis of temperature-sensitive strains (cold-acclimatized strains) (H50-11, L50-33, L50-40) are shown. [Figure 20] This shows the weight fluctuations of hamsters infected with SARS-CoV-2. [Figure 21] This shows the lung weight of a hamster infected with SARS-CoV-2. [Figure 22] This indicates the amount of virus in the lungs or nasal lavage fluid. [Figure 23] This shows the weight fluctuations of hamsters reinfected with SARS-CoV-2. [Figure 24] This shows the neutralizing antibody titer in hamster serum after SARS-CoV-2 infection. [Figure 25] This paper evaluates the neutralizing activity of temperature-sensitive strains (cold-acclimatized strains) against SARS-CoV-2 mutant strains. [Figure 26] This paper compares the immune-inducing ability of temperature-sensitive strains (cold-acclimatized strains) based on the administration route. [Figure 27] This shows a comparison of immune induction ability based on the dosage of temperature-sensitive strains (cold-acclimatized strains). [Figure 28] This paper evaluates the neutralizing activity of temperature-sensitive strains (cold-acclimatized strains) against SARS-CoV-2 mutant strains. [Figure 29] This paper evaluates the neutralizing activity of temperature-sensitive strains (cold-acclimatized strains) against SARS-CoV-2 mutant strains. [Modes for carrying out the invention]

[0013] 1. Beta coronavirus temperature-sensitive strain (cold-acclimatized strain) The betacoronavirus temperature-sensitive strain (cold-acclimatized strain) of the present invention is a betacoronavirus that contains a non-structural protein having a predetermined mutation as the mutation responsible for temperature sensitivity, and is characterized by being temperature-sensitive.

[0014] Morphologically, coronaviruses are spherical, approximately 100-200 nm in diameter, with protrusions on their surface. Virologically, coronaviruses belong to the order Nidovirales, subfamily Coronavirinae, and family Coronaviridae. Within a lipid bilayer envelope is a single-stranded RNA genome wrapped around a nucleocapsid protein (also called a nucleocapsid), and the surface of the envelope is arranged with spike proteins (hereinafter also called "spikes"), envelope proteins (hereinafter also called "envelopes"), and membrane proteins. The viral genome is approximately 30 kb, making it the longest among RNA viruses.

[0015] Coronaviruses are classified into alpha, beta, gamma, and delta groups based on their genetic characteristics. Among coronaviruses that infect humans, four types are known to cause the common cold: human coronavirus 229E, OC43, NL63, and HKU-1. Additionally, the severe acute respiratory syndrome (SARS) coronavirus, which caused severe pneumonia in 2002, and the Middle East respiratory syndrome (MERS) coronavirus, which caused severe pneumonia in 2012, are also known. The alpha coronavirus genus includes human coronavirus 229E and NL63, while the beta coronavirus genus includes human coronavirus OC43, HKU-1, SARS coronavirus, and MERS coronavirus.

[0016] SARS-CoV-2, classified as a SARS coronavirus, was isolated and identified as the causative virus of the novel coronavirus infection that originated in Wuhan in 2019. SARS-CoV-2 has undergone repeated mutations from the initial Wuhan strain, and variant strains have been found in the UK, South Africa, India, and other locations. It is possible that there are still undetected variant strains, and that new variant strains may emerge in the future. In this invention, viruses included in the genus Betacoronavirus are not limited to the above-mentioned SARS-CoV-2 strains, but also include all other Betacoronaviruses (other SARS-CoV-2 variant strains and Betacoronaviruses other than SARS-CoV-2 that may be newly detected in the future).

[0017] The predetermined mutations possessed by the betacoronavirus temperature-sensitive strain (cold-adapted strain) of the present invention will be explained below based on Table 1. Mutations (b), the combination of mutations (e) and (f), and / or mutation (h), indicated as "responsible mutations" in Table 1, are the responsible mutations for temperature sensitivity (cold-adaptation ability) that are essential to the betacoronavirus temperature-sensitive strain (cold-adapted strain) of the present invention. Mutations (a), (c), (d), (g), (i) to (m), indicated as "other mutations" in Table 1, are mutations that the betacoronavirus temperature-sensitive strain (cold-adapted strain) of the present invention may or may not contain at least one of the other mutations.

[0018] [Table 1]

[0019] In other words, the mutations responsible for the temperature sensitivity of the betacoronavirus temperature-sensitive strain (cold-acclimatized strain) of the present invention are the following mutations: (b), a combination of (e) and (f), and / or the mutation (h). (b) A mutation in the amino acid residue corresponding to leucine at position 445 of the amino acid sequence shown in SEQ ID NO: 1 in NSP3, (e) A mutation in the amino acid residue corresponding to glycine at position 248 of the amino acid sequence shown in SEQ ID NO: 2 in NSP14. (f) A mutation in the amino acid residue corresponding to glycine at position 416 of the amino acid sequence shown in SEQ ID NO: 2 in NSP14. (h) A mutation in the amino acid residue corresponding to valine at position 67 of the amino acid sequence shown in SEQ ID NO: 3 in NSP16.

[0020] The betacoronavirus temperature-sensitive strain (cold-acclimatized strain) of the present invention may, in addition to the above-mentioned causative mutation, further include at least one of the following mutations: (a), (c), (d), (g), (i) to (m). (a) A mutation in the amino acid residue corresponding to valine at position 404 of the amino acid sequence shown in SEQ ID NO: 1 in NSP3, (c) Mutation of the amino acid residue corresponding to lysine at position 1792 in the amino acid sequence shown in SEQ ID NO: 1 in NSP3, (d) A mutation in the amino acid residue corresponding to aspartic acid at position 1832 of the amino acid sequence shown in SEQ ID NO: 1 in NSP3, (g) A mutation in the amino acid residue corresponding to alanine at position 504 of the amino acid sequence shown in SEQ ID NO: 2 in NSP14. (i) A mutation in the amino acid residue corresponding to leucine at position 54 of the amino acid sequence shown in SEQ ID NO: 4 in the spike, (j) A mutation in the amino acid residue corresponding to threonine at position 739 of the amino acid sequence shown in SEQ ID NO: 4 in the spike, (k) A mutation in the amino acid residue corresponding to alanine at position 879 of the amino acid sequence shown in SEQ ID NO: 4 in the spike, (l) A mutation in the amino acid residue corresponding to leucine at position 28 of the amino acid sequence shown in SEQ ID NO: 5 in the envelope, and (m) A mutation in the amino acid residue corresponding to the serine at position 2 of the amino acid sequence shown in SEQ ID NO: 6 in the nucleocapsid.

[0021] Sequence ID 1 is the amino acid sequence of NSP3 in SARS-CoV-2 from NC_045512(NCBI); Sequence ID 2 is the amino acid sequence of NSP14 in SARS-CoV-2 from NC_045512(NCBI); Sequence ID 3 is the amino acid sequence of NSP16 in SARS-CoV-2 from NC_045512(NCBI).

[0022] Furthermore, Sequence ID 4 is the amino acid sequence of the spike in SARS-CoV-2 from NC_045512(NCBI); Sequence ID 5 is the amino acid sequence of the envelope in SARS-CoV-2 from NC_045512(NCBI); and Sequence ID 6 is the amino acid sequence of the nucleocapsid in SARS-CoV-2 from NC_045512(NCBI).

[0023] "Corresponding" means that, if the beta-coronavirus temperature-sensitive strain (cold-acclimatized strain) of the present invention is a mutant strain of SARS-CoV-2 of NC_045512 (NCBI), there is a mutation at the predetermined position in the amino acid sequence of SEQ ID NOs. 1-3 or 1-6. If the beta-coronavirus temperature-sensitive strain (cold-acclimatized strain) of the present invention is a beta-coronavirus mutant strain other than the above mutant strain, there is a mutation at the position corresponding to the predetermined position in the amino acid sequence of the polypeptide of the other beta-coronavirus mutant strain corresponding to SEQ ID NOs. 1-3 or 1-6 can be identified by performing amino acid sequence alignment between the protein of SEQ ID NOs. 1-3 or 1-6 of SARS-CoV-2 of NC_045512 (NCBI) and the protein of the other beta-coronavirus mutant strain corresponding to the protein of SEQ ID NOs. 1-3 or 1-6.

[0024] The virus temperature-sensitive strain (cold-acclimatized strain) of the present invention is not limited to specific SARS-CoV-2 mutant strains listed in NC_045512 (NCBI), but includes other beta-coronavirus mutant strains (i.e., any other SARS-CoV-2 mutant strain and mutant strains of viruses other than SARS-CoV-2 included in the genus Beta-coronavirus), as long as the amino acid residue corresponding to the above-mentioned predetermined position in the amino acid sequence of SEQ ID NOs. A specific SARS-CoV-2 mutant strain listed in NC_045512 (NCBI) is a mutant strain in which at least one amino acid residue at the predetermined position in the amino acid sequence represented by SEQ ID NOs: 1-3 or 1-6 of that specific SARS-CoV-2 is mutated. Other beta-coronavirus mutant strains refer to both any other SARS-CoV-2 mutant strain (i.e., a mutant strain in which the amino acid residue corresponding to the predetermined position in the amino acid sequence corresponding to SEQ ID NOs: 1-3 or 1-6 of any other SARS-CoV-2 is mutated) and mutant strains of viruses other than SARS-CoV-2 belonging to the genus Beta-coronavirus (i.e., a mutant strain in which the amino acid residue corresponding to the predetermined position in the amino acid sequence corresponding to SEQ ID NOs: 1-3 or 1-6 of a virus other than SARS-CoV-2 belonging to the genus Beta-coronavirus is mutated).

[0025] The amino acid sequences corresponding to SEQ ID NOs: 1-3 or 1-6 in other betacoronavirus mutant strains may differ from those shown in SEQ ID NOs: 1-3 or 1-6, respectively, as long as the differences do not significantly affect the polypeptide's properties. "Not significantly affecting the polypeptide's properties" means maintaining the respective functions as a non-structural or structural protein. Specifically, differences from SEQ ID NOs: 1-3 or 1-6 are permitted in sites other than the amino acid corresponding to the responsible mutation in SEQ ID NOs: 1-3, or, if there are other mutations, in sites other than the amino acid residues corresponding to the responsible mutation and other mutations in SEQ ID NOs: 1-6 (these non-amino acid sites corresponding to these mutations will also be referred to as "optional difference sites" below). Permitted differences may be one type of difference (e.g., substitution) selected from substitution, addition, insertion, and deletion, or they may include two or more types of differences (e.g., substitution and insertion). The sequence identity calculated by comparing only the arbitrary difference sites between the amino acid sequences corresponding to SEQ ID NOs. 1-3 or 1-6 in any other SARS-CoV-2 and the amino acid sequences shown in SEQ ID NOs. 1-3 or 1-6 should be 50% or more. For any other SARS-CoV-2, the sequence identity is preferably 60% or more, 70% or more, more preferably 80% or more, even more preferably 85% or more, 90% or more, even more preferably 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, and particularly preferably 99.3% or more, 99.5% or more, 99.7% or more, or 99.9% or more. For any remaining beta-coronaviruses, the sequence identity is preferably 60% or more.Here, "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) from BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0026] In other words, the beta-coronavirus temperature-sensitive strain (cold-acclimatized strain) of the present invention is more specifically as follows: Betacoronavirus temperature-sensitive (cold-acclimatized) strains comprising a non-structural protein consisting of at least one of the polypeptides (I), (II), and (III) below: (I) At least one of the following polypeptides (I-1) to (I-3): (I-1) A polypeptide (NSP3) consisting of an amino acid sequence in which the leucine mutation at position 445 (b') is the causal mutation, as shown in SEQ ID NO: 1, (I-2) A polypeptide (NSP14) consisting of an amino acid sequence in which the glycine at position 248 (e') and the glycine at position 416 (f') are the responsible mutations, as shown in SEQ ID NO: 2, (I-3) A polypeptide (NSP16) consisting of an amino acid sequence in which the valine mutation (h') at position 67 is the causal mutation, as shown in SEQ ID NO: 3; (II) A polypeptide constituting a betacoronavirus that has acquired temperature sensitivity (cold acclimation) ability, wherein one or more amino acid residues other than the amino acid residue related to the responsible mutation in the amino acid sequence of the polypeptide in (I) above are substituted, added, inserted, or deleted; (III) A polypeptide constituting a betacoronavirus that has acquired temperature sensitivity (cold acclimation ability), wherein the sequence identity of the amino acid sequence after removing the amino acid residue related to the responsible mutation in the amino acid sequence of the polypeptide in (I) above is 50% or more.

[0027] If the above-mentioned more specific betacoronavirus temperature-sensitive strain (cold-acclimatized strain) includes other mutations in addition to the causative mutation, then the polypeptides (non-structural proteins) of (I-1) and (I-2) above may also be the polypeptides (I-1a) and (I-2a) below, respectively, which have other mutations in addition to the causative mutation, and the polypeptide (I) above may further include the polypeptides (structural proteins) of (I-4a) to (I-6a) below, which have other mutations. A betacoronavirus temperature-sensitive (cold-acclimatized) strain comprising a structural protein, or a structural protein and a non-structural protein, consisting of at least one polypeptide from (I), (II), and (III) below: (I) At least one of (I-1a), (I-2a), (I-3) below, or in addition to those, at least one of (I-4a) to (I-6a) below: (I-1a) A polypeptide (NSP3) having an amino acid sequence in the amino acid sequence shown in Sequence ID No. 1, wherein the responsible mutation is a mutation at leucine at position 445 (b'), and the other mutations are at least one of the following: a mutation at valine at position 404 (a'), a mutation at lysine at position 1792 (c'), and a mutation at aspartic acid at position 1832 (d'), (I-2a) A polypeptide (NSP14) consisting of an amino acid sequence shown in Sequence ID No. 2, in which the responsible mutations are a mutation at glycine position 248 (e') and a mutation at glycine position 416 (f'), and another mutation is a mutation at alanine position 504 (g'), (I-3) A polypeptide (NSP16) consisting of an amino acid sequence in which the responsible mutation is a valine mutation (h') at position 67 in the amino acid sequence shown in Sequence ID No. 3, (I-4a) A polypeptide (spike) consisting of an amino acid sequence in which, in the amino acid sequence shown in Sequence ID No. 4, the other mutations include at least one of the following mutations: a mutation at leucine at position 54 (i'), a mutation at threonine at position 739 (j'), and a mutation at 879 (alanine) (k'). (I-5a) A polypeptide (envelope) consisting of an amino acid sequence in which, as another mutation, a leucine mutation (l') at position 28 is present in the amino acid sequence shown in Sequence ID No. 5, (I-6a) A polypeptide (nucleocapsid) consisting of an amino acid sequence in which, as an additional mutation, a serine mutation (m') at position 2 is present in the amino acid sequence shown in Sequence ID No. 6; (II) A polypeptide constituting a betacoronavirus that has acquired temperature sensitivity (cold accretion) ability, wherein one or more amino acid residues other than those related to the responsible mutation and other mutations are substituted, added, inserted, or deleted in the amino acid sequence of the polypeptide in (I) above; (III) A polypeptide constituting a betacoronavirus that has acquired temperature sensitivity (cold acclimation ability), wherein the sequence identity of the amino acid sequence after removing the amino acid residues related to the responsible mutation and other mutations in the amino acid sequence of the polypeptide in (I) above is 50% or more.

[0028] The mutations (a') to (m') described above refer to mutations that occur when the mutations (a) to (m) specifically exist in the amino acid sequences of SEQ ID NOs. 1 to 6. In other words, the polypeptide (I) described above is a polypeptide consisting of the amino acid sequences of SEQ ID NOs. 1 to 6 possessed by SARS-CoV-2 of NC_045512 (NCBI), into which the responsible mutation, or other mutations in addition, have been introduced. The polypeptides (II) and (III) described above are polypeptides consisting of amino acid sequences corresponding to the amino acid sequences of SEQ ID NOs. 1 to 6 possessed by other beta-coronaviruses, into which the responsible mutation, or other mutations in addition, have been introduced. The preferred range of sequence identity for polypeptides (II) and (III) described above is as previously stated.

[0029] By possessing the above-mentioned responsible mutation, the betacoronavirus can acquire temperature-sensitive (cold-acclimatized) characteristics. The virus temperature-sensitive strain (cold-acclimatized strain) of the present invention has at least a lower ability to proliferate at human lower respiratory tract temperatures than at temperatures lower than human lower respiratory tract temperatures, and preferably does not proliferate at human lower respiratory tract temperatures. In the present invention, temperature sensitivity (cold-acclimatization) ability is determined by comparing, for example, 10% of the viral titer (TCID50 / mL) in the culture supernatant after infecting Vero cells with the virus temperature-sensitive strain at human lower respiratory tract temperatures with an MOI of 0.01 and then culturing them for 1 day at human lower respiratory tract temperatures with the viral titer (TCID50 / mL) in the culture supernatant after infecting Vero cells with the virus temperature-sensitive strain at human upper respiratory tract temperatures with an MOI of 0.01 and then culturing them for 1 day at human upper respiratory tract temperatures. 2 Preferably 10 3 This can be confirmed by the above decrease.

[0030] Typically, the virus temperature-sensitive strain (cold-acclimatized strain) of the present invention exhibits reduced proliferation ability at human lower respiratory tract temperatures compared to the proliferation ability at human lower respiratory tract temperatures in strains without the aforementioned causal mutation. This is because, for example, the viral titer (TCID50 / mL) in the culture supernatant after infecting Vero cells with the virus temperature-sensitive strain at human lower respiratory tract temperatures with an MOI of 0.01 and then culturing them for 1 day is lower than the viral titer in the culture supernatant after infecting Vero cells with a strain without the aforementioned causal mutation at human lower respiratory tract temperatures with an MOI of 0.01 and then culturing them for 1 day. 2 Preferably 10 3 This can be confirmed by the above decrease.

[0031] A typical example of human lower respiratory tract temperature is approximately 37°C, and more specifically, temperatures higher than the upper respiratory tract temperature described later, preferably 36-38°C, more preferably 36.5-37.5°C or 37-38°C. Furthermore, the virus temperature-sensitive strain (low-temperature acclimatized strain) of the present invention may have the ability to proliferate at temperatures below the human lower respiratory tract temperature. For example, temperatures lower than the human lower respiratory tract temperature may include, for example, the human upper respiratory tract temperature (specifically, approximately 32°C-35.5°C).

[0032] The aforementioned responsible mutation is not present on the receptor-binding domain of the spike protein on the viral surface, which is crucial for the virus's infection of cells. Therefore, it is reasonably expected that introducing this responsible mutation can make not only the specific SARS-CoV-2 listed in NC_045512 (NCBI) but also other beta-coronaviruses more temperature-sensitive. In other words, even if a global pandemic causes mutations that alter the immunogenicity of the virus, it is reasonably expected that introducing the aforementioned responsible mutation into the mutant virus can confer temperature sensitivity to that mutant virus.

[0033] Furthermore, regarding the responsible mutations, the mutation in (b) above may be a substitution to an amino acid residue other than leucine, the mutation in (e) above may be a substitution to an amino acid residue other than glycine, the mutation in (f) above may be a substitution to an amino acid residue other than glycine, and the mutation in (h) above may be a substitution to an amino acid residue other than valine. Regarding the other mutations, the mutation in (a) above may be a substitution to an amino acid residue other than valine, the mutation in (c) above may be a substitution to an amino acid residue other than lysine, the mutation in (d) above may be a substitution to an amino acid residue other than aspartic acid, the mutation in (g) above may be a substitution to an amino acid residue other than alanine, the mutation in (i) above may be a substitution to an amino acid residue other than leucine, the mutation in (j) above may be a substitution to an amino acid residue other than threonine, the mutation in (k) above may be a substitution to an amino acid residue other than alanine, the mutation in (l) above may be a substitution to an amino acid residue other than leucine, and the mutation in (m) above may be a substitution to an amino acid residue other than serine.

[0034] In a preferred example of the virus temperature-sensitive strain (cold-acclimatized strain) of the present invention, the responsible mutation is such that the mutation in (b) is a substitution to phenylalanine, the mutation in (e) is a substitution to valine and the mutation in (f) is a substitution to serine, and / or the mutation in (h) is a substitution to isoleucine. If the preferred example further has other mutations, the other mutations are such that the mutation in (a) is a substitution to alanine, the mutation in (c) is a substitution to arginine, the mutation in (d) is a substitution to asparagine, the mutation in (g) is a substitution to valine, the mutation in (i) is a substitution to tryptophan, the mutation in (j) is a substitution to lysine, the mutation in (k) is a substitution to valine, the mutation in (l) is a substitution to proline, and / or the mutation in (m) is a substitution to phenylalanine.

[0035] In another example of the virus temperature-sensitive strain (cold-acclimatized strain) of the present invention, the substitution may be a so-called conservative substitution. This means substitution with an amino acid that has a similar structure and / or properties. For example, examples of conservative substitutions include substitution of a nonpolar amino acid with another nonpolar amino acid if the amino acid before substitution is a non-charged amino acid, substitution of a non-charged amino acid with another non-charged amino acid if the amino acid before substitution is an acidic amino acid with another acidic amino acid, and substitution of a basic amino acid with another basic amino acid if the amino acid before substitution is a basic amino acid. Generally, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan; "non-charged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; "acidic amino acids" include aspartic acid and glutamic acid; and "basic amino acids" include lysine, arginine, and histidine.

[0036] A more preferred example of the virus temperature-sensitive strain (cold-acclimatized strain) of the present invention is the SARS-CoV-2 mutant strain listed in NC_045512 (NCBI), wherein the mutation in (b) (i.e., the mutation in (b')) is a substitution of leucine at position 445 of the amino acid sequence shown in SEQ ID NO: 1 in NSP3 with phenylalanine (L445F); and the mutation in (e) (i.e., the mutation in (e')) is the substitution of the amino acid sequence shown in SEQ ID NO: 2 in NSP14. Examples include a substitution of glycine at position 248 of the acid sequence with valine (G248V), and the mutation in (f) (i.e., the mutation in (f')) being a substitution of glycine at position 416 of the amino acid sequence shown in SEQ ID NO: 2 with serine (G416S) in NSP14; and / or the mutation in (h) (i.e., the mutation in (h')) being a substitution of valine at position 67 of the amino acid sequence shown in SEQ ID NO: 3 with isoleucine (V67I) in NSP16.Examples of cases where the more preferred example also has other mutations include: the mutation in (a) (i.e., the mutation in (a')) is a substitution of valine at position 404 of the amino acid sequence shown in SEQ ID NO: 1 in NSP3 with alanine (V404A); the mutation in (c) (i.e., the mutation in (c')) is a substitution of lysine at position 1792 of the amino acid sequence shown in SEQ ID NO: 1 in NSP3 with arginine (K1792R); the mutation in (d) (i.e., the mutation in (d')) is a substitution of aspartic acid at position 1832 of the amino acid sequence shown in SEQ ID NO: 1 in NSP3 with asparagine (D1832N); the mutation in (g) (i.e., the mutation in (g')) is a substitution of alanine at position 504 of the amino acid sequence shown in SEQ ID NO: 2 in NSP14 with valine (A504V); and the mutation in (i) (i.e., the mutation in (i')) is a substitution of the spike In this case, the mutation (j) (i.e., the (j') mutation) is a substitution of leucine at position 54 of the amino acid sequence shown in SEQ ID NO: 4 with tryptophan (L54W); in the spike, the mutation (j) (i.e., the (j') mutation) is a substitution of threonine at position 739 of the amino acid sequence shown in SEQ ID NO: 4 with lysine (T739K); in the spike, the mutation (k) (i.e., the (k') mutation) is a substitution of alanine at position 879 of the amino acid sequence shown in SEQ ID NO: 4 with valine (A879V); in the envelope, the mutation (l) (i.e., the (l') mutation) is a substitution of leucine at position 28 of the amino acid sequence shown in SEQ ID NO: 5 with proline (L28P); and / or, the mutation (m) (i.e., the (m') mutation) is a substitution of serine at position 2 of the amino acid sequence shown in SEQ ID NO: 6 with phenylalanine (S2F) in the nucleocapsid.

[0037] The betacoronavirus temperature-sensitive (cold-acclimatized) strain of the present invention may further lack the amino acid sequence encoded by the nucleotide sequence shown in Sequence ID No. 7. The nucleotide sequence shown in Sequence ID No. 7 is part of the open reading frame of SARS-CoV-2 in NC_045512 (NCBI).

[0038] Particularly preferred examples of the virus temperature-sensitive strain (cold-acclimatized strain) of the present invention include the following strains. • A strain having the mutation described in (e) above (preferably the mutation described in (e') and / or G248V) and the mutation described in (f) above (preferably the mutation described in (f') and / or G416S) as the responsible mutation; or a strain having the mutation described in (g) above (preferably the mutation described in (g') and / or A504V), the mutation described in (k) above (preferably the mutation described in (k') and / or A879V), the mutation described in (l) above (preferably the mutation described in (l') and / or L28P), and the mutation described in (m) above (preferably the mutation described in (m') and / or S2F) as the responsible mutation. • A strain having the (h) mutation (preferably the (h') mutation and / or V67I) as the responsible mutation; or a strain having any other mutations, such as the (a) mutation (preferably the (a') mutation and / or V404A), the (d) mutation (preferably the (d') mutation and / or D1832N), or the (j) mutation (preferably the (j') mutation and / or T739K); or a strain having a deletion of the amino acid sequence encoded by the nucleotide sequence shown in Sequence ID No. 7. • A strain having the mutation described in (b) above (preferably the mutation described in (b') and / or L445F) as the responsible mutation; or a strain having the mutation described in (c) above (preferably the mutation described in (c') and / or K1792R) as yet another mutation; or a strain having the mutation described in (i) above (preferably the mutation described in (i') and / or L54W); or a strain having a deletion of the amino acid sequence encoded by the nucleotide sequence shown in Sequence ID No. 7.

[0039] 2. Vaccine 2-1. Active ingredients of attenuated vaccines As described above, the beta-coronavirus temperature-sensitive strain (cold-adapted strain) described in "1. Beta-coronavirus temperature-sensitive strain (cold-adapted strain)" can only efficiently proliferate at temperatures lower than the human lower respiratory tract temperature. Therefore, it is expected that it cannot efficiently proliferate at least in the deeper parts of the body, especially in the lower respiratory tract including the lungs, which can cause serious damage, and thus its pathogenicity is significantly reduced. For this reason, the virus temperature-sensitive strain (cold-adapted strain) can be used as a live attenuated vaccine by infecting a living organism with the attenuated virus itself. Accordingly, the present invention also provides a vaccine containing the above-mentioned beta-coronavirus temperature-sensitive strain (cold-adapted strain) as an active ingredient. Details of the active ingredient are as described in "1. Beta-coronavirus temperature-sensitive strain (cold-adapted strain)".

[0040] 2-2. Active ingredients of gene vaccines As described in "1. Beta-coronavirus temperature-sensitive strain (cold-adapted strain)" above, certain mutations contribute to the conferral of temperature sensitivity (cold-adaptation) ability. Therefore, the present invention also provides a beta-coronavirus gene vaccine containing a gene encoding a non-structural protein having the above-mentioned mutation responsible for temperature sensitivity as an active ingredient. Details of the mutation responsible for temperature sensitivity contained in the active ingredient are as described in "1. Beta-coronavirus temperature-sensitive strain (cold-adapted strain)".

[0041] 2-3. Target Viruses The vaccine of the present invention can reasonably be expected to be effective not only against the initial Wuhan strain of the SARS-CoV-2 virus, but also against a wide range of SARS-CoV-2 virus-related strains and viruses other than SARS-CoV-2 that belong to the genus Betacoronavirus, including the variant strain detected in the UK in September 2020 and in South Africa in October 2020, as well as other known variant strains and other unknown variant strains that have not yet been detected. Therefore, the vaccine of the present invention targets Betacoronavirus.

[0042] 2-4. Other ingredients In addition to the active ingredients described above, the vaccine of the present invention may contain other components depending on the purpose and use, such as adjuvants, buffers, isotonic agents, analgesics, preservatives, antioxidants, deodorizers, light-absorbing dyes, stabilizers, carbohydrates, casein digests, and various vitamins.

[0043] Examples of adjuvants include animal oils (such as squalene) or their hydrogenated oils; vegetable oils (such as palm oil and castor oil) or their hydrogenated oils; oily adjuvants containing anhydrous mannitol oleate ester, liquid paraffin, polybutene, caprylic acid, oleic acid, higher fatty acid esters, etc.; water-soluble adjuvants such as PCPP, saponin, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymer, methacrylic acid copolymer, maleic anhydride copolymer, alkenyl derivative polymers, oil-in-water emulsions, and cationic lipids containing quaternary ammonium salts; precipitated adjuvants such as aluminum hydroxide (alum), aluminum phosphate, aluminum sulfate, and aluminum salts or combinations thereof, and sodium hydroxide; microbial toxin components such as cholera toxin and E. coli heat-loopable toxin; and other components (such as bentonite, muramyl dipeptide derivatives, and interleukins).

[0044] Examples of buffering agents include buffer solutions such as phosphates, acetates, carbonates, and citrates. Examples of isotonic agents include sodium chloride, glycerin, and D-mannitol. Examples of pain relievers include benzyl alcohol. Examples of preservatives include thimerosal, parahydroxybenzoic acid esters, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, antibiotics, and synthetic antibacterial agents. Examples of antioxidants include sulfites and ascorbic acid.

[0045] Examples of light-absorbing dyes include riboflavin, adenine, and adenosine. Examples of stabilizers include chelating agents and reducing agents. Examples of carbohydrates include sorbitol, lactose, mannitol, starch, sucrose, glucose, and dextran.

[0046] Furthermore, the vaccine of the present invention may contain one or more other vaccines against viruses or bacteria that cause diseases other than beta-coronavirus infections such as COVID-19. In other words, the vaccine of the present invention may be prepared as a combination vaccine containing other vaccines.

[0047] 2-5. Dosage Forms The dosage form of the vaccine of the present invention is not particularly limited and can be appropriately determined based on the method of administration and storage conditions. Specific examples of dosage forms include liquid formulations and solid formulations, and more specifically, oral formulations such as tablets, capsules, powders, granules, pills, liquid formulations, and syrups; and parenteral formulations such as injections and sprays.

[0048] 2-6. Method of administration The method of administering the vaccine of the present invention is not particularly limited and may include injection (intramuscular, intraperitoneal, intradermal, and subcutaneous), inhalation (through the nasal cavity and oral cavity), and oral administration. Preferably, the vaccine is administered by injection (intramuscular, intradermal, and subcutaneous) and inhalation (through the nasal cavity), and more preferably, through the nasal cavity.

[0049] 2-7. Applicable to The vaccine of the present invention is not particularly limited to any target population that can develop symptoms due to beta-coronavirus infection (preferably a target population that can develop COVID-19 symptoms due to SARS-CoV-2 virus infection), and examples include mammals, more specifically humans; pets such as dogs and cats; and laboratory animals such as mice and hamsters.

[0050] 2-8.Dose The dosage of the vaccine of the present invention is not particularly limited and can be appropriately determined depending on the type of active ingredient, the method of administration, and the target population (conditions such as age, weight, sex, presence or absence of underlying diseases). For example, a dosage of 1 × 10⁻¹⁶ for humans. 10 TCID50 / kg or less, preferably 1 × 10 8 Examples include TCID50 / kg or less.

[0051] 3. Method for producing temperature-sensitive strains (cold-acclimatized strains) of beta-coronavirus The method for producing the betacoronavirus temperature-sensitive strain (cold-acclimatized strain) of the present invention is not particularly limited and can be appropriately determined by those skilled in the art based on the above-described amino acid sequence information. For example, from the viewpoint of producing a vaccine that is relatively inexpensive and has little lot-to-lot variation, a reverse genetics method using an artificial chromosome such as a bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC), or a betacoronavirus genome fragment, is preferred.

[0052] In the reverse genetics method for reconstructing viruses, the genome of a betacoronavirus temperature-sensitive strain (cold-acclimatized strain) that does not contain any of the responsible mutations (parent strain) is first cloned. The parent strain used at this time can be any betacoronavirus, and specifically, it can be selected from the group consisting of a specific SARS-CoV-2 listed in NC_045512 (NCBI) above, any other SARS-CoV-2 listed above, and viruses other than SARS-CoV-2 that belong to the genus Betacoronavirus.

[0053] Furthermore, when using artificial chromosomes in reverse genetics, the full-length DNA of the viral genome is cloned into BAC DNA or YAC DNA, and a transcription promoter sequence for eukaryotic cells is inserted upstream of the viral sequence. Examples of promoter sequences include the CMV promoter and the CAG promoter. A ribozyme sequence and a polyA sequence are inserted downstream of the viral sequence. Examples of ribozyme sequences include the hepatitis D virus ribozyme and the hammerhead ribozyme. Examples of polyA sequences include the polyA of the Simian 40 virus.

[0054] On the other hand, when using the CPER method in reverse genetics, the full-length DNA of the viral genome is divided into multiple fragments and cloned. Methods for obtaining these fragments include artificial nucleic acid synthesis, and PCR using a plasmid cloned from the artificial chromosome or fragment as a template.

[0055] To introduce at least one of the above-mentioned causative mutations into the viral genome cloned by the above method, known point mutation introduction methods such as homologous recombination methods including double crossover and λ / RED recombination, overlap PCR, and CRISPR / Cas9 can be used.

[0056] Next, the artificial chromosome into which the responsible mutation has been introduced is transfected into host cells to reconstruct the recombinant virus. In the reverse genetics method using the CPER method, the fragment into which the responsible mutation has been introduced is ligated using a reaction with DNA polymerase, and then transfected into host cells to reconstruct the recombinant virus. The transfection method is not particularly limited, and known methods can be used. Similarly, the host is not particularly limited, and known cells can be used.

[0057] Next, the reconstituted recombinant virus is added to cultured cells, and the recombinant virus is subcultured. The cultured cells used are not particularly limited, but examples include Vero cells, VeroE6 cells, Vero cells with supplemented TMPRESS2 expression, Calu-3 cells, 293T cells with supplemented ACE2 expression, BHK cells, 104C1 cells, mouse neuroblastoma-derived NA cells, and Vero cells. Virus recovery can be performed by known methods such as centrifugation and membrane filtration. Furthermore, by adding the recovered virus to cultured cells, mass production of recombinant virus becomes possible. [Examples]

[0058] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0059] [Test Example 1] [Test Example 1-1] Isolation of SARS-CoV-2 temperature-sensitive strain (cold-acclimatized strain) A50-18. Based on the method shown in Figure 1, two mutagenic agents, 5-fluorouracil (hereinafter referred to as strain B-1) and 5-azacytidine (hereinafter referred to as 5-AZA), were added to a clinical isolate of SARS-CoV-2 to obtain virus populations of the A-F50 series and A-F500 series, which were acclimatized at 32°C. Furthermore, each virus population was passed through multiple cycles, and from the resulting 406 candidate strains, a virus strain (A50-18 strain; sometimes referred to as the Ts strain hereafter) that could grow at 32°C but whose growth rate was significantly reduced at 37°C was identified, isolated, and selected (Figure 2).

[0060] [Test Example 1-2] Analysis of temperature-sensitive strains (cold-acclimatized strains) A50-18 using next-generation sequencing. (1-2-1) Mutation analysis of each virus strain Mutation analysis of the following virus strains was performed using next-generation sequencing. This analysis was carried out by extracting RNA from the culture supernatant of Vero cells infected with SARS-CoV-2. Wuhan-Hu-1 (NC045512), a clinical isolate from Wuhan, was used as the reference. B-1: Wild strain (clinical isolate) A50-18: Temperature sensitive strain (low temperature acclimated strain) F50-37: non-temperature sensitive strain C500-1: non-temperature sensitive strain F500-53: non-temperature sensitive strain F500-40: non-temperature sensitive strain F500-2: non-temperature sensitive strain (All except B-1 are mutated viruses)

[0061] (1-2-2) Mutation of temperature-sensitive strains (cold-acclimatized strains) The analysis results shown in Figure 3A were obtained from (1-2-1). The point mutation D614G is also found in strain B-1, so it is not a point mutation characteristic of temperature-sensitive strains (cold-acclimatized strains). On the other hand, characteristic point mutations of temperature-sensitive strains (cold-acclimatized strains) (A50-18) were found to be G248V, G416S, A504V of NSP14, A879V of Spike, L28P of Envelope, and S2F of Nucleocapsid.

[0062] (1-2-3) Analysis of Revertant Mutants 1 A "revert mutation" refers to a return to the same phenotype as the original virus before the mutation, due to further mutations in a mutated virus. In this specification, a "revert mutation" refers to the loss of temperature sensitivity in a temperature-sensitive strain due to further mutations. Further mutations include the return of the amino acids at the mutated site to the pre-mutation amino acids.

[0063] Vero cells were infected with strain B-1 or strain A50-18 at MOI=0.01, and their growth was evaluated at 32°C, 34°C, and 37°C. Among the A50-18 strains, a sample with restored growth at 37°C (hereinafter referred to as the "revertor mutant") was found. In the revertor mutant, it appears that some amino acid residues in the temperature-sensitive strain (A50-18 strain) that acquired temperature sensitivity underwent a reversion mutation (hereinafter simply referred to as "revertor mutation") back to the pre-mutation amino acid, resulting in decreased temperature sensitivity and restored growth at 37°C. Figure 3B shows a CPE image illustrating this. Sequence analysis of the obtained samples revealed that the G248V mutation in NSP14 had reverted to the wild-type G, while the G416S, A504V, and L28P mutations in NSP14 and the envelope were maintained. This suggests that the G248V mutation in NSP14 is the responsible mutation contributing to temperature sensitivity.

[0064] (1-2-4) Analysis of reverse mutations in isolates 2 The A50-18 strain was used to infect Vero cells with moi=1, and its proliferation was evaluated at 37°C and 38°C. As a result, samples (reverted mutants) with restored proliferation at 37°C and 38°C were found. Figure 3C shows the CPE image of the obtained reverted mutants after culturing at 37°C for 3 days. The sequence of the obtained reverted mutants was confirmed as follows: <1> and <2> Two types of recovery patterns were identified. <1> In NSP14, the G248V mutation reverts to the wild-type G, while the G416S and A504V mutations remain. <2> In NSP14, the G416S mutation reverts to the wild-type G, while the G248V and A504V mutations are maintained.

[0065] Since a revert mutation in at least one of the G248V and G416S mutations in NSP14 resulted in loss of temperature sensitivity, it was suggested that the combination of the G248V and G416S mutations in NSP14 is the responsible mutation contributing to temperature sensitivity.

[0066] (1-2-5) Analysis of recombinant viruses in which mutations have been introduced into the wild type BAC DNA containing the entire wild-type SARS-CoV-2 genome was introduced by homologous recombination with NSP14, Spike, Nucleocapsid, and Envelope from strain A50-18. The resulting recombinant BAC DNA was transfected into 293T cells to reconstitute the virus. The recombinant virus was used to infect Vero cells, and temperature sensitivity was evaluated by observing CPE at 37°C and 32°C. The results are shown in Figure 3D. By introducing NSP14 from strain A50-18, a temperature-sensitive strain that did not show CPE at 37°C culture was obtained, indicating that NSP14 is the causative mutation contributing to temperature sensitivity. On the other hand, introducing Envelope from strain A50-18 did not result in temperature sensitivity, suggesting that the Envelope mutation does not contribute to temperature sensitivity.

[0067] (1-2-6) Analysis of recombinant viruses with mutations introduced into the wild type 2 The virus was reconstructed using the CPER method, introducing mutations in NSP14. Three types of recombinant viruses were reconstructed, each possessing one of the following NSP14 mutations. • G248V only • G416S only • G248V and G416S (hereinafter also referred to as "double mutants"). Each recombinant virus was used to infect Vero cells, and CPE was observed after incubation at 37°C or 32°C for 3 days. As shown in Figure 3-E, viruses with only the G248V mutation and viruses with only the G416S mutation showed CPE at both 37°C and 32°C, similar to strain B-1, indicating that they were not temperature-sensitive. On the other hand, in the double mutant virus with both the G248V and G416S mutations, CPE was observed at 32°C, but only slightly at 37°C, indicating significantly weaker CPE compared to 32°C. From these results, it became clear that the presence of the G248V and G416S mutations slows down viral replication at 37°C, making the virus temperature-sensitive. From this, it was found that the combination of the G248V and G416S mutations in NSP14 is the responsible mutation contributing to temperature sensitivity.

[0068] (1-2-6) Analysis of temperature-sensitive strains (cold-acclimatized strains) by Sanger sequencing Mutation analysis of strain A50-18 was performed using Sanger sequencing. This analysis was carried out by extracting RNA from the culture supernatant of SARS-CoV-2 infected Vero cells. As a result, no deletions like those observed in (2-2-5) of Test Example 2-2 described later were found.

[0069] (1-2-7) Summary of mutations in temperature-sensitive strains (cold-acclimatized strains) In the temperature-sensitive strain (cold-acclimatized strain) A50-18, as shown in Table 2 below, mutations marked with a checkmark were found in the amino acid sequence of the indicated sequence number, and among these, the mutation marked with a double checkmark was identified as the causative mutation. Furthermore, as shown in Table 2 below, a double mutant strain possessing only the causative mutation in NSP14 was also found to be a temperature-sensitive strain (cold-acclimatized strain).

[0070] [Table 2]

[0071] [Test Example 1-3] Analysis of Proliferation of Temperature-Sensitive Strains (Cold-Acclimated Strains) A50-18 (1-3-1) Analysis at 32°C and 37°C Clinical isolates (B-1 strain) and temperature-sensitive strains (cold-acclimatized strains) (A50-18 strain) were used to infect Vero cells in a 6-well plate under MOI = 0.01 or 0.1 conditions (N=3). After culturing at 37°C or 32°C, the culture supernatant was collected at 0–5 dpi. The viral titer of the 0–5 dpi culture supernatant was measured using Vero cells at TCID 50 / mL. The results are shown in Figure 4A. Figure 4A shows that strain A50-18 had a viral titer below the detection limit on day 3 after infection at 37°C, indicating a significant decrease in its ability to proliferate at 37°C.

[0072] (1-3-2) Analysis at 32°C, 34°C, and 37°C Clinical isolates (B-1 strain) and temperature-sensitive strains (cold-acclimatized strains) (A50-18 strain) were used to infect Vero cells in a 6-well plate under MOI = 0.01 conditions (N=3). After culturing at 37°C, 34°C, or 32°C, the culture supernatant was collected at 0–5 dpi. The viral titer of the 0–5 dpi culture supernatant was measured using Vero cells at TCID 50 / mL. The results are shown in Figure 4B. Figure 4B shows that strain A50-18 proliferated at 32°C and 34°C to a similar extent as the clinical isolate, but exhibited a significant lack of proliferative capacity at 37°C.

[0073] [Test Example 1-4] Pathogenicity analysis of temperature-sensitive strain (cold-acclimatized strain) A50-18 (1-4-1) Weight fluctuations in hamsters infected with SARS-CoV-2 Four 4-week-old male Syrian hamsters (n=4) were reared for one week, and then clinical isolates (B-1 strain) and temperature-sensitive strains (cold-acclimatized strains) (A50-18 strain) (1x10 4 or 1x10 6 TCID50) was administered intranasally at a volume of 100 μL, and body weight changes were observed for 10 days. A group administered the same volume of D-MEM medium intranasally served as a non-infected control (MOCK). The results are shown in Figure 5. No weight loss was observed when infected with strain A50-18, suggesting significantly low pathogenicity.

[0074] (1-4-2) Viral load in the lungs or nasal cavities of SARS-CoV-2-infected hamsters After male Syrian hamsters at 4 weeks of age (n = 3) were raised for one week, a clinical isolate (B-1 strain) and a temperature-sensitive strain (cold-adapted strain) (A50-18 strain) (1x10 6 TCID50) was administered intranasally in a volume of 100 μL. The results of observing the body weight changes over 3 days are shown in Fig. 6. After the hamsters were euthanized at 3 dpi, nasal lavage fluid was collected with 1 mL of D-PBS. Also, the lungs of the hamsters were excised, the right lung was disrupted, suspended in 1 mL of D-MEM, and the supernatant was collected as a lung disruption fluid by centrifugation. The results of evaluating the viral load in these nasal lavage fluids and lung disruption fluids using a plaque formation assay with Vero cells are shown in Fig. 7. Furthermore, the excised left lung was fixed with 10% formalin and photographed, and the result is shown in Fig. 8.

[0075] From Fig. 7, it was found that there was no difference in the viral load between the B-1 strain and the A50-18 strain in the nasal lavage fluid, while the virus of the A50-18 strain was significantly less in the lungs. Also, from Fig. 8, it was found that in hamsters infected with the B-1 strain and showing weight loss, swelling and blackening were observed in the lungs, while in hamsters infected with the A50-18 strain, no significant changes in body weight or lungs were observed. From the above results, it is presumed that the temperature-sensitive strain (cold-adapted strain) is a weakly virulent strain that proliferates in the upper respiratory tract but cannot proliferate in the lower respiratory tract.

[0076] (1-4-3) Histological analysis of SARS-CoV-2-infected hamsters Sections were prepared from the formalin-fixed lungs obtained from the infection experiment on hamsters performed in (1-4-2) and stained with HE to analyze the histological pathogenicity of the lungs due to SARS-CoV-2 infection. The results are shown in Fig. 9.

[0077] As shown in Figure 9, in the lungs of hamsters infected with the clinical isolate (B-1 strain), red blood cell infiltration and alveolar structure collapse were observed. On the other hand, these severe symptoms were not observed in the lungs of hamsters infected with the temperature-sensitive strain (cold-acclimatized strain) (A50-18 strain). This strongly suggests that the A50-18 strain does not induce severe inflammation in the lungs upon infection and has low pathogenicity.

[0078] (1-4-4) Histological analysis of SARS-CoV-2 infected hamsters by immunochemical staining Regarding the histological pathogenicity observed in (1-4-3), viral proteins were detected by immunochemical staining to evaluate the relationship between viral replication and pathogenicity. Four-week-old male Syrian hamsters (B-1, A50-18: n=5, MOCK: n=3) were reared for one week, and then the clinical isolate (B-1 strain) and the temperature-sensitive strain (cold-acclimatized strain) (A50-18 strain) (1x10) were tested. 6 TCID50) was administered intranasally at a volume of 100 μL. After euthanasia at 3 dpi, the excised left lung was fixed in 10% formalin and serial sections were prepared. The obtained serial sections were subjected to HE staining and immunochemical staining (also known as IHC staining). Rabbit anti-spike polyclonal antibody (Sinobiological: 40589-T62) was used for immunochemical staining. The HE stained and immunochemical stained images are shown in Figure 10. Similar to (1-4-3), hamsters infected with strain B-1 showed erythrocyte infiltration and breakdown of alveolar structure, and spike protein was detected over a wide area by immunochemical staining. On the other hand, hamsters infected with strain A50-18 did not show such tissue damage, and spike protein was detected only in locally limited areas. These results clearly show that strain B-1 exhibits significant viral proliferation and tissue damage in lung tissue, while strain A50-18 is unable to efficiently proliferate in lung tissue and exhibits low levels of lung tissue damage.

[0079] [Test Examples 1-5] Immunogenicity analysis of temperature-sensitive strains (cold-acclimatized strains) A50-18 (1-5-1) Test to attack wild-type strains on hamsters infected with temperature-sensitive strains (cold-acclimatized strains) The following procedure was used to conduct an attack test with wild-type strains (clinical isolates) on hamsters infected with temperature-sensitive strains (cold-acclimatized strains). Four 4-week-old male Syrian hamsters (n=4) were reared for one week, then clinical isolates (B-1 strain) or temperature-sensitive strains (cold-acclimatized strains) (A50-18 strain) (1x10 4 or 1x10 6 TCID50) was administered intranasally at a volume of 100 μL. 21 days later, the clinical isolate (B-1 strain) (1x10) was administered again. 6 TCID50) was administered intranasally at a volume of 100 μL, and weight changes were observed for 10 days. Three hamsters of the same age that had never been infected (n=3) were used as naive controls. The results are shown in Figure 11.

[0080] As shown in Figure 11, naive hamsters showed weight loss after infection with strain B-1, while hamsters that had been infected once with strain B-1 or A50-18 did not experience weight loss. This indicates that immunity contributing to infection defense can be induced not only by the wild-type strain B-1, but also by infection with the less pathogenic A50-18 strain.

[0081] (1-5-2) Analysis of neutralizing antibody induction in hamsters infected with temperature-sensitive strains (cold-acclimatized strains) After raising 4-week-old male Syrian hamsters (n=5) for one week, they were given either a clinical isolate (B-1 strain) or a temperature-sensitive strain (cold-acclimatized strain) (A50-18 strain) (1x10 6 TCID50) was administered intranasally at a volume of 100 μL. Post-infection weight changes are shown in Figure 12. As with previous results, weight loss was observed with infection with strain B-1, while no weight loss was observed with infection with strain A50-18.

[0082] Twenty-one days after infection, whole blood was collected, serum was separated, and the serum was inactivated by heat at 56°C for 30 minutes. 100 TCID50 B-1 strains were mixed with serially diluted inactivated serum and reacted at 37°C for 1 hour. The culture medium after the reaction was seeded into Vero cells, incubated at 37°C, and the neutralizing activity of the virus was evaluated by observing CPE. The highest dilution ratio that did not produce CPE was defined as the neutralizing antibody titer. The results are shown in Figure 13. It was revealed that serum from uninfected hamsters did not show neutralizing activity against the B-1 strain, while serum from hamsters infected with the B-1 or A50-18 strains was able to induce neutralizing antibodies.

[0083] [Test Example 2] [Test Example 2-1] Additional isolation of SARS-CoV-2 temperature-sensitive strains (cold-acclimated strains) H50-11, L50-33, and L50-40 strains To isolate further candidate strains, temperature-sensitive strains (cold-acclimatized strains) were isolated using the method shown in Figure 14. Clinical isolates of SARS-CoV-2 (hereinafter referred to as strain B-1) were used to infect Vero cells, and the G-L50 series of viruses were obtained by acclimatizing them at 32°C with the addition of the mutagenesis agent 5-FU. Furthermore, each virus population was passed through multiple times, and from the 253 strains obtained, virus strains that could grow at 32°C but whose growth rate was significantly reduced at 37°C (strains H50-11, L50-33, and L50-40) were identified, isolated, and selected (Figure 15).

[0084] [Test Example 2-2] Analysis of additional temperature-sensitive (cold-acclimatized) isolates H50-11, L50-33, and L50-40 using next-generation sequencing. (2-2-1) Method for analyzing mutations in additional isolates Mutation analysis of the following virus strains was performed using next-generation sequencing. This analysis was carried out by extracting RNA from the culture supernatant of Vero cells infected with SARS-CoV-2. Wuhan-Hu-1 (NC045512), a clinical isolate from Wuhan, was used as the reference. H50-11 strain: Temperature sensitive strain (low temperature acclimated strain) L50-33 strain: Temperature sensitive strain (low temperature acclimated strain) L50-40 strain: Temperature sensitive strain (low temperature acclimated strain)

[0085] (2-2-2) Results of mutation analysis of temperature-sensitive strains (cold-acclimatized strains) In (2-2-1), the analysis results shown in Figure 16A were obtained. Characteristic point mutations of strain H50-11 were found to be V404A and D1832N of NSP3, V67I of NSP16, and T739K of Spike. Characteristic point mutations of strain L50-33 were found to be L445F and K1792R of NSP3, and characteristic point mutations of strain L50-40 were found to be L445F and K1792R of NSP3 and L54W of Spike.

[0086] (2-2-3) Analysis of revertant mutants of additional isolates (H50-11 strain) The H50-11 strain was used to infect Vero cells with moi=1, and its proliferation was evaluated at 37°C and 38°C. As a result, samples (revertor mutants) in which proliferation at 37°C and 38°C was restored were found. Figure 16B shows the CPE image of the obtained revertor mutants after being cultured at 38°C for 3 days. Sequencing of the obtained samples revealed that the NSP16 V67I mutation had reverted to wild-type V, while other amino acid mutations were maintained. This suggests that the NSP16 V67I mutation is the causative mutation contributing to temperature sensitivity.

[0087] (2-2-4) Analysis of revertant mutants of additional isolates (L50-33 and L50-40) L50-33 and L50-40 strains were used to infect Vero cells with an MOI of 0.01, and their proliferation was evaluated at 32°C, 34°C, and 37°C. As a result, samples with restored proliferation at 37°C (hereinafter referred to as revertant mutants) were found among the L50-33 and L50-40 strains. Figure 16C shows the CPE images of each strain after 3 days of culture at 37°C. The revertant mutants of L50-33 and L50-40 strains are referred to as L50-33 strain Rev1 and 2, and L50-40 strain Rev1 and 2, respectively. Sequencing of the obtained samples revealed that the L445F mutation in NSP3 had mutated to wild-type L or C, while the K1792R mutation in NSP3 was maintained. This suggests that the L445F mutation in NSP3 may be the causative mutation contributing to temperature sensitivity.

[0088] (2-2-5) Analysis of temperature-sensitive strains (cold-acclimatized strains) by Sanger sequencing Mutation analysis of strains H50-11, L50-33, and L50-40 was performed using Sanger sequencing. This analysis was carried out by extracting RNA from the culture supernatant of SARS-CoV-2 infected Vero cells.

[0089] As a result, a deletion in the nucleotide sequence (SEQ ID NO: 7) at positions 27549-28251 was found in all three strains, as shown in Figure 17. A schematic diagram of the deletion in the nucleotide sequence at positions 27549-28251 and the corresponding amino acid sequence deletion is shown in Figure 18. In Figure 18, ORF7a is the nucleotide sequence at positions 27394-27759, ORF7b is the nucleotide sequence at positions 27756-27887, and ORF8 is the nucleotide sequence at positions 27894-28259.

[0090] As shown in Figure 18, the nucleotide sequence region from position 27549 to 28251 corresponds to a portion of ORF7a (the amino acid sequence from position 53 to the terminal; the same applies hereafter), the entirety of ORF7b, and most of the amino acid sequence of ORF8. Since deletion of this region is accompanied by a frameshift, it is thought that a protein will be produced in which the amino acid sequence of ORF7a from position 1 to 52 is fused with the amino acid sequence encoded by the 3' terminal 8 nucleotides, the intergenetic region, and the nucleocapsid nucleotide sequence of ORF8. Furthermore, ORF7b will be completely deleted, and the original sequence of ORF8 will also be completely deleted.

[0091] (2-2-6) Summary of mutations in temperature-sensitive strains (cold-acclimatized strains) In the temperature-sensitive strains (cold-acclimatized strains) H50-11, L50-33, and L50-40, as shown in Table 3 below, mutations marked with a checkmark were found in the amino acid sequences of the indicated sequence numbers, and among these, the mutations marked with a double checkmark were identified as the causative mutations.

[0092] [Table 3]

[0093] [Test Example 3] Proliferative activity analysis of additional isolates H50-11, L50-33, and L50-40 Additional isolates were used to infect Vero cells under MOI=0.01 conditions (N=3). After culturing at 37°C, 34°C, or 32°C, the culture supernatant was collected at 0–5 dpi. The viral titers of these culture supernatants were measured using TCID. 50 Measurements were taken using Vero cells at / mL. The results are shown in Figure 19. The resulting additional isolates showed proliferation at 32°C and 34°C, while their proliferation was delayed and decreased at 37°C.

[0094] [Test Example 4] Pathogenicity analysis of each temperature-sensitive strain (4-1) Weight fluctuations in hamsters infected with temperature-sensitive strains After raising 4-week-old male Syrian hamsters (n=5) for one week, clinical isolates (B-1 strain) or temperature-sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, H50-11 strain) (3x105 TCID50) was administered intranasally at a volume of 100 μL, and body weight changes were observed for 10 days. The group administered the same volume of D-MEM medium intranasally was used as the non-infected control (MOCK). The results are shown in Figure 20. Hamsters infected with strain B-1 showed a weight loss of approximately 20% over 7 days, while hamsters infected with the temperature-sensitive strain showed no significant weight loss in any group, suggesting significantly lower pathogenicity.

[0095] (4-2) Viral load in the lungs or nasal cavity of hamsters infected with temperature-sensitive strains After raising 4-week-old male Syrian hamsters (n=5) for one week, clinical isolates (B-1 strain) or temperature-sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, H50-11 strain) (3x10 5 TCID50) was administered intranasally at a volume of 100 μL. After euthanasia at 3 dpi, the nasal lavage fluid was collected with 1 mL of D-PBS. The hamsters' lungs were also excised, their weight measured, and the right lung was lysed. After suspension in 1 mL of D-MEM, the supernatant was collected as lung lysate by centrifugation. The lung weight per unit of the hamster's total body weight is shown in Figure 21. The viral load in these nasal lavage fluids and lung lysates was evaluated using a plaque formation assay with Vero cells, and the results are shown in Figure 22.

[0096] A comparison of lung weight per unit of total body weight in hamsters revealed that hamsters infected with strain B-1 showed increased lung weight, strongly suggesting lung swelling due to inflammation. Conversely, this increase in lung weight was not observed in hamsters infected with temperature-sensitive strains. Furthermore, a comparison of viral loads in nasal lavage fluid showed no significant difference between hamsters infected with strain B-1 and those infected with temperature-sensitive strains (excluding strain H50-11), with hamsters infected with strain H50-11 showing lower viral loads in nasal lavage fluid. Additionally, hamsters infected with temperature-sensitive strains exhibited significantly lower intrapulmonary viral loads than hamsters infected with strain B-1. These results suggest that each temperature-sensitive strain, like strain A50-18 in Test Example 1, is an attenuated strain unable to replicate in the lower respiratory tract.

[0097] [Test Example 5] Immunogenicity analysis of each temperature-sensitive strain (5-1) Wild-type strain attack test on hamsters infected with temperature-sensitive strain After raising 4-week-old male Syrian hamsters (n=5) for one week, clinical isolates (B-1 strain) or temperature-sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, H50-11 strain) (3x10 5 TCID50) was administered intranasally at a volume of 100 μL. 21 days later, the clinical isolate (B-1 strain) (3 x 10) was administered again. 5 TCID50) was administered intranasally at a volume of 100 μL, and body weight changes were observed for 9 days. Five hamsters of the same age that had never been infected before (n=5) were used as naive controls. The results are shown in Figure 23. Naive hamsters showed weight loss after infection with strain B-1, while hamsters that had been infected once with strain B-1 or each temperature-sensitive strain did not experience weight loss. This indicates that immunity contributing to infection defense can be induced not only by the wild-type strain B-1, but also by infection with each temperature-sensitive strain with low pathogenicity.

[0098] (5-2) Analysis of neutralizing antibody induction in hamsters infected with temperature-sensitive strains After raising 4-week-old male Syrian hamsters (n=5) for one week, clinical isolates (B-1 strain) or temperature-sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, H50-11 strain) (3x10 5 TCID50) was administered intranasally in a volume of 100 μL. Partial blood samples were taken 20 days later, and the neutralizing activity against the clinical isolate (B-1 strain) was measured using the obtained serum. The same method as in (1-5-2) was used to measure the neutralizing activity. The measurement results are shown in Figure 24. It was revealed that antibodies with neutralizing activity were induced not only in hamsters infected with the B-1 strain, but also in hamsters infected with each temperature-sensitive strain.

[0099] [Test Example 6] Efficacy Analysis against SARS-CoV-2 Mutants (6-1) Evaluation of the neutralizing activity of temperature-sensitive strain infected hamster serum against SARS-CoV-2 mutants After rearing 4-week-old male Syrian hamsters (n=3 or 5) for one week, they were given either a clinical isolate (B-1 strain) or a temperature-sensitive strain (A50-18 strain) (3x10 5 TCID50) was administered intranasally at a volume of 100 μL. Partial blood samples were taken from hamsters three weeks after infection, and the neutralizing activity against live SARS-CoV-2 European clinical isolate (B-1) and Brazilian variant strain (hCoV-19 / Japan / TY7-503 / 2021) was measured using the obtained serum. The results are shown in Figure 25. The method for measuring neutralizing activity was the same as in (1-5-2). It was revealed that hamsters infected with the B-1 strain and temperature-sensitive strain also showed neutralizing activity against the Brazilian variant strain. From this, it can be considered that this attenuated live vaccine may also be effective against SARS-CoV-2 variant strains.

[0100] [Test Example 7] Comparative study of administration routes and dosages (7-1) Comparison of immune induction ability by administration route After raising 4-week-old male Syrian hamsters (n=5) for one week, they were given either a clinical isolate (B-1 strain) or a temperature-sensitive strain (A50-18 strain) (3x10 5 TCID50) was administered intranasally or subcutaneously at a volume of 100 μL. The untreated group was designated as the naive control. Three weeks later, the neutralizing activity against the SARS-CoV-2 Brazilian variant (hCoV-19 / Japan / TY7-503 / 2021 strain) was evaluated using serum obtained by partial blood sampling from hamsters. The same method as in (1-5-2) was used to measure neutralizing activity. The results of the neutralizing activity are shown in Figure 26. "in" indicates intranasal administration, and "SC" indicates subcutaneous administration. Intranasal administration of strains B-1 and A50-18 induced neutralizing antibodies against live viruses. Subcutaneous administration did not induce neutralizing antibodies at the tested doses, but considering the results for intranasal administration, it was thought that increasing the dose would induce neutralizing antibodies even with subcutaneous administration.

[0101] (7-2) Comparison of immune induction ability by dosage Five four-week-old male Syrian hamsters were reared for one week, after which the temperature-sensitive strain (A50-18 strain) was administered intranasally or subcutaneously. The dosage is shown in Table 4.

[0102] [Table 4] Figure 27 shows the results of measuring the neutralizing activity against live SARS-CoV-2 Brazilian variant (hCoV-19 / Japan / TY7-503 / 2021 strain) using serum obtained from partial blood samples taken from hamsters three weeks after infection. 'in' indicates intranasal administration, and 'SC' indicates subcutaneous administration. The method for measuring neutralizing activity was the same as in (1-5-2). Similar to (7-1), 1 x 10⁻⁶ for intranasal administration. 2 An increase in neutralizing antibody titers was observed even in the low-dose TCID50 / 10 μL administration group. This suggests that temperature-sensitive strains may be able to induce sufficient immunity even with small amounts of intranasal administration. Regarding subcutaneous administration, the tested doses did not induce much neutralizing antibody, but considering the results from intranasal administration, it is thought that neutralizing antibodies can be induced even with subcutaneous administration if the dose is increased.

[0103] [Test Example 8] Efficacy Analysis against SARS-CoV-2 Mutants After raising 4-week-old male Syrian hamsters (n=4) for one week, 1x10 4 TCID50 or 1x10 2 A temperature-sensitive strain of TCID50 (A50-18 strain) was administered intranasally at a volume of 10 μL. Partial blood samples were taken from hamsters three weeks after infection, and the neutralizing activity against live SARS-CoV-2 European wild-type strain (B-1 strain), Indian mutant strain (autoisotope), and Brazilian mutant strain (hCoV-19 / Japan / TY7-503 / 2021 strain) was measured using the obtained serum. The results are shown in Figure 28. The method for measuring neutralizing activity was the same as in (1-5-2). It was revealed that even in individuals that received a small amount of A50-18 intranasally, neutralizing antibodies against not only the parental wild-type B-1 strain, but also the Indian and Brazilian mutant strains could be induced in a dose-dependent manner.

[0104] Furthermore, Figure 29 shows the results of comparing the neutralizing antibody titers in the serum of each individual against each strain. Although a decrease in neutralizing antibody titers was observed in some individuals against the Brazilian mutant strain, it was clear that all individuals possessed neutralizing antibodies. These results suggest that immunization by intranasal administration of the temperature-sensitive strain may exhibit cross-protection.

Claims

1. A betacoronavirus temperature-sensitive strain comprising a non-structural protein having the following combination of mutations (e) and (f) as the mutation responsible for temperature sensitivity, wherein the betacoronavirus is SARS coronavirus: (e) Substitution of the amino acid residue corresponding to glycine at position 248 of the amino acid sequence shown in Sequence ID No. 2 in NSP14 with valine, (f) Substitution of the amino acid residue corresponding to glycine at position 416 of the amino acid sequence shown in Sequence ID No. 2 in NSP14 with serine.

2. The virus temperature-sensitive strain according to claim 1, wherein the SARS coronavirus is the SARS-CoV-2 virus.

3. The virus temperature-sensitive strain according to claim 1 or 2, wherein its ability to proliferate at human lower respiratory tract temperatures is reduced compared to the proliferative ability of a betacoronavirus containing a non-structural protein that does not have the aforementioned causal mutation.

4. The virus temperature-sensitive strain according to claim 3, wherein the human lower respiratory tract temperature is 36 to 38°C.

5. The virus temperature-sensitive strain according to claim 1, wherein the non-structural protein further has the following mutations: (g) Substitution of the amino acid residue corresponding to alanine at position 504 of the amino acid sequence shown in Sequence ID No. 2 in NSP14 with valine.

6. The aforementioned non-structural protein further has the following mutation (g), A virus temperature-sensitive strain according to claim 1 or 2, comprising a structural protein having the following mutation (k), the following mutation (l), and the following mutation (m): (g) A mutation in the amino acid residue corresponding to alanine at position 504 of the amino acid sequence shown in Sequence ID No. 2 in NSP14, which is a substitution with valine. (k) A mutation in the amino acid residue corresponding to alanine at position 879 of the amino acid sequence shown in SEQ ID NO: 4 in the spike, which is a substitution with valine. (l) A mutation in the envelope, which is the amino acid residue corresponding to leucine at position 28 of the amino acid sequence shown in SEQ ID NO: 5, and is a substitution with proline. (m) A mutation in the nucleocapsid, specifically the amino acid residue corresponding to the serine at position 2 of the amino acid sequence shown in SEQ ID NO: 6, which is replaced with phenylalanine.

7. A live attenuated vaccine comprising a virus temperature-sensitive strain according to any one of claims 1 to 6.

8. A live attenuated vaccine according to claim 7, administered intranasally.

9. A betacoronavirus gene vaccine comprising a gene encoding a non-structural protein having the following combination of mutations (e) and (f) as the mutation responsible for temperature sensitivity, wherein the betacoronavirus is SARS coronavirus: (e) Substitution of the amino acid residue corresponding to glycine at position 248 of the amino acid sequence shown in Sequence ID No. 2 in NSP14 with valine, (f) Substitution of the amino acid residue corresponding to glycine at position 416 of the amino acid sequence shown in Sequence ID No. 2 in NSP14 with serine.

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